Acid production by fermentation

ABSTRACT

The invention provides methods for producing Lactate by anaerobic Fermentation. According to particular methods of the invention, Lactate is produced by anaerobic fermentation of a substrate comprising hydrogen and carbon monoxide.

FIELD OF THE INVENTION

This invention relates to the production of lactate by microbialfermentation of substrates comprising CO.

BACKGROUND OF THE INVENTION

Lactic acid is an important platform chemical with many applications.Over the last decade, lactic acid has gained importance in thedetergence industry. Lactic acid has descaling as well as anti-bacterialproperties, so has been used as an environmentally beneficial cleaningproduct. Furthermore, lactic acid is a precursor for severalbiodegradable polymers such as polylactic acid. These types of plasticsprovide a good option for substituting conventional plastics producedfrom petroleum oil because of low CO2 emissions. Other applicationsinclude precursors for lactate esters, which can replace petrochemicalderived solvents.

Lactic acid is typically produced by fermentation of carbohydrates suchas glucose, fructose and sucrose. The most commercially important genusof lactic acid fermenting bacteria is Lactobacillus, though otherbacteria and even yeast are also used. In such fermentations, lacticacid is formed through the reduction of pyruvate, which is in turnproduced by glycolysis. The cost of these carbohydrate feed stocks isinfluenced by their value as human food or animal feed. For example,cultivation of starch or sucrose-producing crops for lactic acidproduction is not economically sustainable in all geographies.Therefore, it is of interest to develop technologies to convert lowercost and/or more abundant carbon resources into lactic acid.

Carbon Monoxide (CO) is a major by-product of the incomplete combustionof organic materials such as coal or oil and oil derived products.Although the complete combustion of carbon containing precursors yieldsCO2 and water as the only end products, some industrial processes needelevated temperatures favouring the build up of carbon monoxide overCO2. One example is the steel industry, where high temperatures areneeded to generate desired steel qualities. For example, the steelindustry in Australia is reported to produce and release into theatmosphere over 500,000 tonnes of CO annually.

Furthermore, CO is also a major component of syngas, where varyingamounts of CO and H2 are generated by gasification of acarbon-containing fuel. For example, syngas may be produced by crackingthe organic biomass of waste woods and timber to generate precursors forthe production of fuels and more complex chemicals.

The release of CO into the atmosphere may have significant environmentalimpact. In addition, emissions taxes may be required to be paid,increasing costs to industrial plants. Since CO is a reactive energyrich molecule, it can be used as a precursor compound for the productionof a variety of chemicals. However, this valuable feedstock has not beenutilised to produce lactic acid.

It is an object of the present invention to provide a process that goesat least some way towards overcoming the above disadvantages or at leastto provide the public with a useful choice.

STATEMENT OF INVENTION

In one aspect, the invention provides a method of producing lactic acidby microbial fermentation of a substrate comprising carbon monoxide. Inparticular embodiments, the invention provides a method of producinglactic acid by microbial fermentation, the method including:

-   -   a. providing a substrate comprising CO;    -   b. in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate to        produce lactic acid.

In particular embodiments, at least 0.05 g/day lactic acid per Litre offermentation broth is produced. In particular embodiments, at least 0.1g/L/day; or at least 0.2 g/L/day; or at least 0.3 g/L/day; or at least0.5 g/L/day; or at least 1.0 g/L/day lactic is produced.

In another aspect, the invention provides a method of increasingefficiency lactic acid production by fermentation, the method including:

-   -   a. providing a substrate comprising CO;    -   b. in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate to        produce lactic acid.

In another aspect of the invention, there is provided a method ofproducing lactic acid by microbial fermentation, the method including:

-   -   a. providing a substrate    -   b. in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate, wherein        one or more micro-organisms includes one or more lactate        dehydrogenase genes;    -   c. up-regulating the lactate dehydrogenase gene(s), such that        lactic acid is produced by the micro-organism(s).

In particular embodiments, the substrate comprises CO.

In particular embodiments of the various aspects, the substratecomprising carbon monoxide is a gaseous substrate comprising carbonmonoxide. The gaseous substrate comprising carbon monoxide can beobtained as a by-product of an industrial process. In certainembodiments, the industrial process is selected from the groupconsisting of ferrous metal products manufacturing, non-ferrous productsmanufacturing, petroleum refining processes, gasification of biomass,gasification of coal, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing. In one embodiment the gaseous substrate comprises a gasobtained from a steel mill. In another embodiment the gaseous substratecomprises automobile exhaust fumes.

In particular embodiments, the CO-containing substrate typicallycontains a major proportion of CO, such as at least about 20% to about100% CO by volume, from 40% to 95% CO by volume, from 40% to 60% CO byvolume, and from 45% to 55% CO by volume. In particular embodiments, thesubstrate comprises about 25%, or about 30%, or about 35%, or about 40%,or about 45%, or about 50% CO, or about 55% CO, or about 60% CO byvolume. Substrates having lower concentrations of CO, such as 6%, mayalso be appropriate, particularly when H₂ and CO₂ are also present.

In particular embodiments of the various aspects, the substratecomprising CO is provided at a sufficient level, such that lactic acidis produced. In particular embodiments, CO is provided such that aspecific uptake rate of at least 0.4 mmol/g/min; or at least 0.5mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; orat least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min ismaintained.

In certain embodiments of the various aspects, the method comprisesmicrobial fermentation using one or more carboxydotrophic microorganismsvia the Wood-Ljungdahl pathway. In particular embodiments, themicroorganism is a clostridia, such as Clostridium autoethanogenum.

In another aspect, the invention provides a method of producing lacticacid by microbial fermentation, the method including:

-   -   a. providing a substrate    -   b. in a bioreactor including a culture of Clostridium        autoethanogenum, anaerobically fermenting the substrate to        produce lactic acid.

In particular embodiments, the substrate is one or more carbohydratessuch as fructose. Alternatively the substrate is a substrate comprisingcarbon monoxide, more preferably a gaseous substrate comprising carbonmonoxide, as herein before described

In a further aspect of the invention, there is provided a methodaccording to any of the previous aspects, wherein the method furtherincludes the step of capturing or recovering the lactic acid.

In a further aspect, there is provided lactic acid produced by themethods of any of the previous aspects.

The invention may also be said broadly to consist in the parts, elementsand features referred to or indicated in the specification of theapplication, individually or collectively, in any or all combinations oftwo or more of said parts, elements or features, and where specificintegers are mentioned herein which have known equivalents in the art towhich the invention relates, such known equivalents are deemed to beincorporated herein as if individually set forth.

FIGURES

FIG. 1 is graph demonstrating lactate production according a method ofthe invention as described in example 1.

FIG. 2 is a graph demonstrating lactate production according a method ofthe invention as described in example 2.

FIG. 3 is a graph demonstrating lactate production according a method ofthe invention as described in example 3.

FIG. 4 is a graph demonstrating lactate production according a method ofthe invention as described in example 4.

FIG. 5 a, FIG. 5 b, and FIG. 5 c are graphs showing the effects ofdiffering lactate concentrations on cell growth and metaboliteproduction as described in example 5. The figure legend on FIG. 5 c alsorelates to FIGS. 5 a and 5 b.

DETAILED DESCRIPTION OF THE INVENTION

The following is a description of the present invention, includingpreferred embodiments thereof, given in general terms. The invention isfurther exemplified in the disclosure given under the heading “Examples”herein below, which provides experimental data supporting the invention,specific examples of aspects of the invention, and means of performingthe invention.

The terms ‘lactic acid’ and ‘lactate’ have been used hereininterchangeably and include all stereoisomers of 2-hydroxy propionicacid including (R), (S) and racemic forms.

The term “bioreactor” includes a fermentation device consisting of oneor more vessels and/or towers or piping arrangement, which includes theContinuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR),Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, StaticMixer, or other vessel or other device suitable for gas-liquid contact.As is described herein after, in some embodiments the bioreactor maycomprise a first growth reactor and a second fermentation reactor. Assuch, when referring to the addition of a substrate, for example asubstrate comprising carbon monoxide, to the bioreactor or fermentationreaction it should be understood to include addition to either or bothof these reactors where appropriate.

The term “substrate comprising carbon monoxide” and like terms should beunderstood to include any substrate in which carbon monoxide isavailable to one or more strains of bacteria for growth and/orfermentation, for example.

“Gaseous substrates comprising carbon monoxide” include any gas whichcontains a level of carbon monoxide. The gaseous substrate willtypically contain a major proportion of CO, preferably at least about15% to about 95% CO by volume.

Unless the context requires otherwise, the phrases “fermenting”,“fermentation process” or “fermentation reaction” and the like, as usedherein, are intended to encompass both the growth phase and productbiosynthesis phase of the process.

The inventors have surprisingly shown that lactic acid can be producedby microbial fermentation of a substrate comprising CO. It has beensurprisingly shown that lactic acid can be produced by carboxydotrophicbacteria by fermentation of a substrate comprising CO. The inventorshave found that fermentation produces several products whereby ethanoland lactic acid are significant substituents. Lactic acid has not beenpreviously identified as a product of fermentation of a substratecomprising CO. In accordance with the methods of the invention, it hasalso been surprisingly demonstrated that lactic acid can be produced byClostridium autoethanogenum from substrates comprising CO, particularlygaseous substrates comprising CO. The use of a gaseous carbon source,particularly a source including CO, in fermentation processes has notpreviously resulted in the production of lactic acid.

In particular embodiments of the invention, the efficiency of lacticacid production can be increased by providing the substrate at asufficient level such that lactic acid is produced. It has beenrecognised that increasing the amount of substrate provided to amicrobial culture, increases the amount of lactic acid produced by theculture.

In particular embodiments of the invention, the substrate comprising COis provided at a sufficient level such that lactic acid is produced. Ithas been shown that a microbial culture comprising C. autoethanogenumcan uptake CO at a rate up to approximately 1.0 to 2 mmol/gram dryweight microbial cells/minute (specific CO uptake). In particularembodiments of the invention, a substrate comprising CO is provided tothe microbial culture comprising C. autoethanogenum such that a specificuptake is maintained substantially at or at least 0.4 mmol/g/min; or atleast 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min. In such embodiments, lactic acid is a significantfermentation product of at least 0.05 g/L; or at least 0.1 g/L; or atleast 0.2 g/L; or at least 0.3 g/L; or at least 0.4 g/L; or at least 0.5g/L. In particular embodiments, lactic acid is produced at a rate of atleast 0.5 g/L/day; or at least 1 g/L/day.

In particular embodiments of the invention, apparatus used forconducting methods of the invention enable measurement and/or control ofparameters such as CO supply, CO uptake, biomass level, pH, lactic acidproduction. For example, samples can be taken from a bioreactor todetermine one or more of the above parameters and the bioreactorconditions optionally adjusted to improve lactic acid production. Forexample, in a bioreactor, wherein the microbial culture is producing noor insignificant amounts of lactic acid, the CO supply can be increasedsuch that lactic acid is produced.

It is accepted that products such as acetate and ethanol are producedfrom CO via a combination of the acetyl-CoA cycle and the THF cycle viathe Wood-Ljungdahl pathway as described in Phillips, J. R, et al, 1994,Applied Biochemistry and Biotechnology, 45/46: 145. However, inaccordance with the methods of the invention, it has been surprisinglyshown that lactic acid can be produced, particularly where CO isprovided such that specific CO uptake rates of at least 0.4 mmol/g/min;or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min are maintained. Without wishing to be bound by theory, it isconsidered that by providing sufficient or elevated levels of CO, higherenergy products, such as lactic acid can be produced duringfermentation. It is considered precursors of products, such as lacticacid act as electron acceptors to relieve the microbial cell of excessreducing power, in the form of NAD(P)H, thus restoring a favourableNAD(P):NAD(P)H equilibrium. It is further considered that carbohydratesfermented by the culture can also be converted into lactic acid in asimilar manner.

A putative NAD-dependent D-(−)-lactate dehydrogenase gene with a lengthof 981 bases could be identified in the genome sequence of C.autoethanogenum LZ1560 (strain deposited at DSMZ under the accessionnumber 19630). The monocistronic gene shows high homology (74% identity(486/656), E-value 2e-55) to the partial D-(−)-lactate dehydrogenasegene IdhA of Clostridium sp. strains IBUN 13A (Accession Nr.GQ_(—)180219.1) and IBUN 158B (Accession Nr. GQ_(—)180219.1).

Nucleotide sequence:ATGAAAGTTTTGGCATATAGTCATAGACAAGATGAAACTGAATATTTCAAAAAATTCAGTAAAAAATACGACGTGGAGGTTGTATTGTGTGATGATCCACCAACTATGGAAAATGCAGACTTGGCCAAGGGATTTGACTGCATCAGCATTATCACAACTAAAATTTCAGATAAATTAGTAGAAAAATTTCATGAAATTGGAGTAAAATTTATATCTACAAGAACAATAGGATATGACCATATAGACATAAAAAAGGCAAAAGAGCTAGGTGTCCATATAGGCAATGTAAACTATTCACCAAATAGTGTAGCCGATTATACAATTATGATGATTCTTATGGCTATAAGAAAAACGAAAGCTATTATAGAACGAAGTAATGTACAGGATTATTCTTTAAAAGGTGTTCAAGGTAAAGAGCTTCACAATTTAACTGTAGGTGTTATTGGTACAGGAAGAATTGGCCGTGCAGTTATAAGTCGCTTAAGTGGATTTGGCTGCAAAATATTAGCTTATGATTTATATGAGAATGAAGAAATAAAGAAGTATGTTACATATGTTACACTAGAAGATCTCTTTAAAAACAGTGACATTATTACAATGCATGCACCTGCAACAGATGACAATTATCACATGATAAATAAGGATTCCATAGCACTTATGAAAGATGGTACATTTATTATCAATATAGCCCGAGGCTCACTTATCAATACTGAAGATCTTATAGATGCCATTGAAAATAAAAAAATTGGTGGTGCAGCTATAGACGTTATTGAAAATGAATTCGGACTTTGCTATAACGATTTAAAATGTGAGATACTAGATAAAAGGGAAATGGCAATTTTAAAATCTTTTCCAAATGTAATTGTAACACCTCACACAGCTTTTTATACAGATCAAGCTGTAAGTGATATGGTAGAACATTCTATTTTAAGTTGTGTTTTATTCATGGAAGGCAAAGAAAATCCATGGCAAATTGAATAA

The gene encodes a protein of 321 amino acids, which has high homologyto alpha keto acid dehydrogenases of other Clostridial species (seetable 1), such as the lactate dehydrogenase of C. acetobutylicum whichhas exactly the same length. A COG (Clusters of Orthologous Groups ofproteins) analysis assigns the protein to the functional group of‘lactate dehdrogenases and related dehydrogenases’ (COG1052).

Protein sequence:MKVLAYSHRQDETEYFKKFSKKYDVEVVLCDDPPTMENADLAKGFDCISIITTKISDKLVEKFHEIGVKFISTRTIGYDHIDIKKAKELGVHIGNVNYSPNSVADYTIMMILMAIRKTKAIIERSNVQDYSLKGVQGKELHNLTVGVIGTGRIGRAVISRLSGFGCKILAYDLYENEEIKKYVTYVTLEDLFKNSDIITMHAPATDDNYHMINKDSIALMKDGTFIINIARGSLINTEDLIDAIENKKIGGAAIDVIENEFGLCYNDLKCEILDKREMAIL KSFPNVIVTPHTAFYTDQAVSDMVEHSILSCVLFMEGKENPWQIE*

Further evidence comes from a domain search carried out against PROSITE(Hulo N, et al, (2008) ‘The 20 years of PROSITE’ Nucleic Acids Res 36:D245-249) and Pfam (Finn et al (2010) ‘The Pfam protein familiesdatabase’ Nucleic Acids Res 38: D211-222) databases. The PROSITE searchyielded two strong hits for a ‘D-isomer specific 2-hydroxyaciddehydrogenases NAD-binding signature’ (PS00065 and PS00671), while a‘D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain’ and a‘D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain’could be identified from the Pfam search with a high E-value of 8.1e-72and 2.1e-30, respectively.

The protein is proposed to catalyze the reaction pyruvate+NADH toD-(−)-lactate+NAD⁺ according to other NAD dependent D-(−)-lactatedehydrogenases (EC 1.1.1.28).

TABLE 1 Best protein BLAST (Altschul et al., (1990) ‘Basic localalignment search tool’ J Mol Biol 215: 403-410) hits for lactatedehydrogenase of Clostridium autoethanogenum LZ1560 DescriptionAccession Nr. Identity E-value D-isomer specific 2-hydroxyacidYP_001308638.1 73% 1e−143 dehydrogenase, NAD-binding (241/326)[Clostridium beijerinckii NCIMB 8052] alpha-keto acid dehydrogenaseYP_001395598.1 71% 1e−141 [Clostridium kluyveri DSM 555] (226/325)lactate dehydrogenase NP_348170.1 69% 1e−137 [Clostridium acetobutylicum(226/325) ATCC 824] D-specific alpha-keto acid ZP_02948009.1 67% 1e−130dehydrogenase (220/325) [Clostridium butyricum 5521]

It is considered lactate dehydrogenase can be upregulated in accordancewith the methods of the invention. For example, where CO is supplied atsufficient levels, lactate dehydrogenase is upregulated. In particular,where CO is supplied such that the specific CO uptake by the microbialculture is at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or atleast 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; orat least 1.2 mmol/g/min; or at least 1.5 mmol/g/min; lactatedehydrogenase is upregulated. As such, the invention provides a methodof producing lactate by microbial fermentation of a substrate byupregulation of lactate dehydrogenase.

It is considered that products such as lactate can be produced by theWood-Ljungdahl pathway in carboxydotrophic micro-organisms such asClostridium autoethanogenum by fermentation of alternative substratessuch as carbohydrates. Thus, in particular embodiments, the methodincludes fermentation of substrates comprising carbohydrate, such asfructose or xylose, to produce products including lactic acid.

It is further considered that alternative substrates, such as acarbohydrate substrate and a gaseous substrate comprising CO, can beswitched during microbial production of lactic acid, without deleteriouseffect. In addition, it is contemplated that substrates could bealternated, for example when one substrate is unavailable, the alternatesubstrate is provided such that the micro-organism continues to producelactic acid.

In accordance with the results obtained, in one embodiment of theinvention, lactic acid is produced by microbial fermentation of asubstrate comprising carbohydrate. In another embodiment of theinvention, a substrate comprising carbon monoxide, preferably a gaseoussubstrate comprising CO, is converted into various products includinglactic acid, by Clostridium autoethanogenum.

It is contemplated that the lactic acid produced in accordance with themethods of the invention may be readily recovered using separationtechniques known in the art.

The invention is generally described herein in relation to preferredembodiments of the invention which utilise Clostridium autoethanogenumand/or produce lactic acid. However, it should be appreciated thatalternative micro-organisms may be substituted for C. autoethanogenumsuch as alternative micro-organisms which ferment substrates comprisingCO, for example C. ljungdahlii, C. ragsdalei and C. carboxydivorans.Other carboxydotrophic microorganisms that produce products via the WoodLjungdahl pathway may also be used.

Method

In one embodiment, the invention provides a method for the production oflactic acid by microbial fermentation. In a preferred embodiment themethod comprises at least the step of anaerobically fermenting asubstrate comprising CO, preferably a gaseous substrate comprising CO,to obtain lactic acid.

In a particular embodiment of the invention, the method includes thesteps of:

-   -   (a) providing a substrate comprising CO, preferably a gaseous        substrate comprising CO;    -   (b) in a bioreactor containing a culture of one or more        micro-organisms anaerobically fermenting the substrate to        produce lactic acid.

In another embodiment, the invention provides a method of increasingefficiency of lactic acid production by fermentation, the methodincluding:

-   -   (a) providing a substrate comprising CO;    -   (b) in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate to        produce lactic acid.

In particular embodiments, the substrate comprising CO is provided at alevel sufficient to produce significant amounts of lactic acid, such asat least 0.05 g/L of fermentation media, or at least 0.1 g/L, or atleast 0.2 g/L, or at least 0.4 g/L, or at least 0.6 g/L, or at least 0.8g/L, or at least 1 g/L. In certain embodiments, CO is provided at alevel sufficient to produce lactic acid at a rate of at least 0.5g/L/day; or at least 1 g/L/day. In particular embodiments, CO isprovided such that a specific uptake rate of at least 0.4 mmol/g/min; orat least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min is maintained.

Those skilled in the art will appreciate methods of supplying CO,particularly gaseous CO, such that the required uptake rate is achieved.However, by way of example, factors such as increasing gas hold-up in afermentation media will increase the amount of CO available forconversion to products by the microbial culture. Gas hold-up cantypically be increased by mechanical means, such as increasing agitationin a CSTR. Furthermore, supplying CO at a faster rate or a higherpartial pressure will also increase the CO availability in afermentation broth.

In another embodiment, the method involves fermentation of a substratecomprising carbohydrate by Clostridium autoethanogenum to produce lacticacid.

In certain embodiments of the invention, the method further includes thestep of capturing or recovering the lactic acid produced.

Micro-Organisms

In embodiments of the invention the one or more micro-organisms used inthe fermentation is one or more carboxydotrophic micro-organisms. Inparticular embodiments, the microorganisms are Clostridia, such asClostridium autoethanogenum. In particular embodiments, themicro-organism ferments a substrate comprising CO via the Wood-Ljungdahlpathway. In a preferred embodiment the Clostridium autoethanogenum is aClostridium autoethanogenum having the identifying characteristics ofthe strain deposited at the German Resource Centre for BiologicalMaterial (DSMZ) under the identifying deposit number 19630. In anotherembodiment the Clostridium autoethanogenum is a Clostridiumautoethanogenum having the identifying characteristics of DSMZ depositnumber DSMZ 10061.

Culturing of the bacteria used in a method of the invention may beconducted using any number of processes known in the art for culturingand fermenting substrates using anaerobic bacteria. Exemplary techniquesare provided in the “Examples” section of this document. By way offurther example, those processes generally described in the followingarticles using gaseous substrates for fermentation may be utilised: K.T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1991).Bioreactors for synthesis gas fermentations resources. Conservation andRecycling, 5; 145-165; K. T. Klasson, M. D. Ackerson, E. C. Clausen andJ. L. Gaddy (1991). Bioreactor design for synthesis gas fermentations.Fuel. 70. 605-614; K. T. Klasson, M. D. Ackerson, E. C. Clausen and J.L. Gaddy (1992). Bioconversion of synthesis gas into liquid or gaseousfuels. Enzyme and Microbial Technology. 14; 602-608; J. L. Vega, G. M.Antorrena, E. C. Clausen and J. L. Gaddy (1989). Study of GaseousSubstrate Fermentation: Carbon Monoxide Conversion to Acetate. 2.Continuous Culture. Biotech. Bioeng. 34. 6. 785-793; J. L. Vega, E. C.Clausen and J. L. Gaddy (1989). Study of gaseous substratefermentations: Carbon monoxide conversion to acetate. 1. Batch culture.Biotechnology and Bioengineering. 34. 6. 774-784; and, J. L. Vega, E. C.Clausen and J. L. Gaddy (1990). Design of Bioreactors for Coal SynthesisGas Fermentations. Resources, Conservation and Recycling. 3. 149-160.Methods for culturing bacteria on substrates comprising carbohydratesare also well known in the art.

Substrates

In one embodiment of the invention, lactic acid is produced by microbialfermentation of a substrate comprising carbohydrate using Clostridiumautoethanogenum. It will be appreciated there are many examples ofcarbohydrates suitable for fermentation known in the art and manyexamples of the types of processes used to ferment the carbohydratesubstrate. By way of example, suitable substrates may include, but arenot limited to, monosaccharides such as glucose and fructose,oligosaccharides such as sucrose or lactose, polysaccharides, such ascellulose or starch. Although it is contemplated that any of thesecarbohydrate substrates (and mixtures thereof) are suitable in thepresent invention, preferred carbohydrate substrates are fructose andsucrose (and mixtures thereof).

Those skilled in the art will appreciate fermentable sugars may beobtained from cellulosic and lignocellulosic biomass through processesof pre-treatment and saccharification, as described, for example, inUS20070031918. Biomass refers to any cellulose or lignocellulosicmaterial and includes materials comprising cellulose, and optionallyfurther comprising hemicellulose, lignin, starch, oligosaccharidesand/or monosaccharides. Biomass includes, but is not limited tobioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, sludge from paper manufacture, yard waste, woodand forestry waste. However, in exemplary embodiments of the inventioncommercially available fructose is used as the carbon and energy sourcefor the fermentation.

In a particular embodiment, a substrate comprising carbon monoxide,preferably a gaseous substrate comprising carbon monoxide is used in themethods of the invention. The gaseous substrate may be a waste gasobtained as a by-product of an industrial process, or from some othersource such as from combustion engine (for example automobile) exhaustfumes. In certain embodiments, the industrial process is selected fromthe group consisting of ferrous metal products manufacturing, such as asteel mill, non-ferrous products manufacturing, petroleum refiningprocesses, gasification of coal, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing. In these embodiments, the CO-containing gas may becaptured from the industrial process before it is emitted into theatmosphere, using any convenient method. Depending on the composition ofthe gaseous substrate comprising carbon monoxide, it may also bedesirable to treat it to remove any undesired impurities, such as dustparticles before introducing it to the fermentation. For example, thegaseous substrate may be filtered or scrubbed using known methods.

In other embodiments of the invention, the gaseous substrate comprisingcarbon monoxide may be sourced from the gasification of biomass. Theprocess of gasification involves partial combustion of biomass in arestricted supply of air or oxygen. The resultant gas typicallycomprises mainly CO and H₂, with minimal volumes of CO₂, methane,ethylene and ethane. For example, biomass by-products obtained duringthe extraction and processing of foodstuffs such as sugar fromsugarcane, or starch from maize or grains, or non-food biomass wastegenerated by the forestry industry may be gasified to produce aCO-containing gas suitable for use in the present invention.

The CO-containing substrate will typically contain a major proportion ofCO, such as at least about 20% to about 100% CO by volume, from 40% to95% CO by volume, from 40% to 60% CO by volume, and from 45% to 55% COby volume. In particular embodiments, the substrate comprises about 25%,or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO,or about 55% CO, or about 60% CO by volume. Substrates having lowerconcentrations of CO, such as 6%, may also be appropriate, particularlywhen H₂ and CO₂ are also present.

In particular embodiments, CO is supplied at a level sufficient forlactic acid production to occur. In particular embodiments, CO isprovided such that a specific uptake rate of at least 0.4 mmol/g/min; orat least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min is maintained.

Those skilled in the art will appreciate methods of supplying CO,particularly gaseous CO, such that the required uptake rate is achieved.Higher rates of CO uptake can be achieved by improving mass transfer ofthe system, wherein the amount of CO dissolved in a typically aqueousfermentation broth increases. However, by way of example, factors suchas increasing gas hold-up in a fermentation media will increase theamount of CO available for conversion to products by the microbialculture. Those skilled in the art will appreciate methods of increasinggas hold-up. However, by way of non-limiting example, gas hold-up istypically increased by mechanical means such as increasing agitation ina CSTR. Furthermore, supplying CO at a faster rate or a higher partialpressure will also increase the CO availability in a fermentation broth.

It is not necessary for the gaseous substrate to contain any hydrogen,however this is not considered detrimental to lactic acid production.The gaseous substrate may also contain some CO₂ for example, such asabout 1% to about 80% by volume, or 1% to about 30% by volume. In oneembodiment it contains about 5% to about 10% by volume. In anotherembodiment the gaseous substrate contains approximately 20% CO₂ byvolume.

Typically, the carbon monoxide will be added to the fermentationreaction in a gaseous state. However, the invention should not beconsidered to be limited to addition of the substrate in this state. Forexample, the carbon monoxide could be provided in a liquid. For example,a liquid may be saturated with a carbon monoxide containing gas and thenthat liquid added to a bioreactor. This may be achieved using standardmethodology. By way of example a microbubble dispersion generator(Hensirisak et. al. Scale-up of microbubble dispersion generator foraerobic fermentation; Applied Biochemistry and Biotechnology Volume 101,Number 3/October, 2002) could be used.

Media

It will be appreciated that for growth of the bacteria and substrate tolactate fermentation to occur, in addition to the substrate, a suitablenutrient medium will need to be fed to the bioreactor. A nutrient mediumwill contain components, such as vitamins and minerals, sufficient topermit growth of the micro-organism used. Anaerobic media suitable forthe growth of carboxydotrophic Clostridia such as Clostridiumautoethanogenum are known in the art, as described for example by Abriniet al (Clostridium autoethanogenum, sp. Nov., An Anaerobic BacteriumThat Produces Ethanol From Carbon Monoxide; Arch. Microbiol., 161:345-351(1994)). The “Examples” section herein after provides furtherexamples of suitable media.

Fermentation Conditions

The fermentation should desirably be carried out under appropriateconditions for the substrate to lactic acid fermentation to occur.Reaction conditions that should be considered include temperature, mediaflow rate, pH, media redox potential, agitation rate (if using acontinuous stirred tank reactor), inoculum level, maximum substrateconcentrations and rates of introduction of the substrate to thebioreactor to ensure that substrate level does not become limiting, andmaximum product concentrations to avoid product inhibition. Examples offermentation conditions suitable for anaerobic fermentation of asubstrate comprising CO are detailed in WO2007/117157, WO2008/115080,WO2009/022925 and WO2009/064200, the disclosure of which areincorporated herein by reference. It is recognised the fermentationconditions reported therein can be readily modified in accordance withthe methods of the instant invention.

The inventors have determined that, in one embodiment where pH is notcontrolled, there does not appear to be a deleterious effect on lacticacid production.

Bioreactor

Fermentation reactions may be carried out in any suitable bioreactor asdescribed previously herein. In some embodiments of the invention, thebioreactor may comprise a first, growth reactor in which themicro-organisms are cultured, and a second, fermentation reactor, towhich broth from the growth reactor is fed and in which most of thefermentation product (lactic acid, for example) is produced.

Product Recovery

The fermentation will result in fermentation broth comprising lactateand, possibly, one or more by-products, such as ethanol or acetate, aswell as bacterial cells in a liquid nutrient media. Lactate or lacticacid can be removed from the typically aqueous fermentation broth by anyknown method. For example, conventional fermentation process producescalcium lactate precipitate, which can be collect and re-acidified.

Alternatively, membrane techniques, such as electrodialysis can be suedto separate lactate. Low concentrations of lactate can be separated froma fermentation broth by applying a suitable potential across a selectiveion permeable membrane. Other suitable techniques includenanofiltration, wherein monovalent ions can selectively pass through amembrane under pressure.

Other desirable products, such as acetate and/or ethanol can be removedform the fermentation broth using any recovery means known in the art,such as methods described in WO2007/117157, which is fully incorporatedherein by reference.

The invention will now be described in more detail with reference to thefollowing non-limiting examples.

EXAMPLES Materials and Methods

Solution A NH₄Ac 3.083 g KCl 0.15 g MgCl₂•6H₂O 0.61 g NaCl 0.12 gCaCl₂•2H₂O 0.294 g Distilled Water Up to 1 L Quantity/ Quantity/Component/0.1M ml into Component/0.1M ml into solution (aq) 1 L mediasolution (aq) 1 L media Solution(s) B FeCl₃ 1 ml Na₂WO₄ 0.1 ml CoCl₂ 0.5ml ZnCl₂ 0.1 ml NiCl₂ 0.5 ml Na₂MoO₄ 0.1 ml H₃BO₃ 0.1 ml Solution CBiotin 20.0 mg Calcium D-(*)- 50.0 mg pantothenate Folic acid 20.0 mgVitamin B12 50.0 mg Pyridoxine•HCl 10.0 mg p-Aminobenzoic acid 50.0 mgThiamine•HCl 50.0 mg Thioctic acid 50.0 mg Riboflavin 50.0 mg Distilledwater To 1 Litre Nicotinic acid 50.0 mg Solution D Solution A 50 mlSolution C 10 ml Solution B 10xconc Distilled Water Up to 1 L Na₂S_(x) 2ml Solution E Solution B 10xconc Na₂S_(x) 2 ml Solution F NH₄Ac 0.5 gKCl 0.15 g MgCl₂•6H₂O 0.5 g NaCl 0.2 g CaCl₂•2H₂O 0.26 g NaH₂PO₄ 2.04 gSolution G 10 ml Solution H 10 ml Resazurin 1 ml FeCL₃ (5 g/L stock) 2ml (2 g/L stock) Cysteine HCL 0.5 g Agar optional Distilled Water Up to1 L Solution G - Composite Mineral Stock Solution Nitrolotriacetic 1.5 gMgSO₄•7H₂O 3.0 g acid MnSO₄•H₂O 0.5 g NaCl 1.0 g FeSO₄•7H₂O 0.1 gFe(SO₄)₂(NH₄)₂•6H₂O 0.8 g CoCl₂•6H₂O 0.2 g ZnSO₄•7H₂O 0.2 g CuCl₂•2H₂O0.02 g AlK(SO₄)₂•12H₂O 0.02 g H₃BO₃ 0.30 g NaMoO₄•2H₂O 0.03 g *Na₂SeO₃0.02 g *NiCl₂•6H₂O 0.02 g Na₂WO₄•6H₂O 0.02 g Distilled Water Up to 1 LSolution H - Genthner B-Vitamin Solution Thiamine 50.0 mg Riboflavin50.0 mg hydrochloride (Vitamin B2) (Vitamin B1) Nicotinic acid 50.0 mgPantothenic acid 50.0 mg (Niacin or (Vitamin B5) Vitamin B3) Pyridoxine10.0 mg Biotin (Vitamin B7) 20.0 mg hydrochloride (Vitamin B6) Folicacid 20.0 mg 4-Aminobenzoic acid 50.0 mg (Vitamin B9) (PABA or VitaminB10) Cyanocobalamin 50.0 mg Distilled water To 1 Litre (Vitamin B12)Lipoic acid 50.0 mg (Thioctic acid)

Preparation of Na₂S_(x)

A 500 ml flask was charged with Na₂S (93.7 g, 0.39 mol) and 200 ml H₂O.The solution was stirred until the salt had dissolved and sulfur (25 g,0.1 mol) was added under constant N₂ flow. After 2 hours stirring atroom temperature, the “Na₂S_(x)” solution (approx 4M with respect to[Na] and approx 5M with respect to [S]), now a clear reddish brownliquid, was transferred into N₂ purged serum bottles, wrapped inaluminium foil.

Preparation of Cr (II) Solution

A 1 L three necked flask was fitted with a gas tight inlet and outlet toallow working under inert gas and subsequent transfer of the desiredproduct into a suitable storage flask. The flask was charged withCrCl₃.6H₂O (40 g, 0.15 mol), zinc granules [20 mesh] (18.3 g, 0.28 mol),mercury (13.55 g, 1 mL, 0.0676 mol) and 500 mL of distilled water.Following flushing with N₂ for one hour, the mixture was warmed to about80° C. to initiate the reaction. Following two hours of stirring under aconstant N₂ flow, the mixture was cooled to room temperature andcontinuously stirred for another 48 hours by which time the reactionmixture had turned to a deep blue solution. The solution was transferredinto N₂ purged serum bottles and stored in the fridge for future use.

Bacteria:

In a preferred embodiment the Clostridium autoethanogenum is aClostridium autoethanogenum having the identifying characteristics ofthe strain deposited at the German Resource Centre for BiologicalMaterial (DSMZ) under the identifying deposit number 10061. In anotherembodiment the Clostridium autoethanogenum is a Clostridiumautoethanogenum having the identifying characteristics of DSMZ depositnumber DSMZ 23693.

Sampling and Analytical Procedures

Media samples were taken from the CSTR reactor at intervals over periodsup to 10 days. Each time the media was sampled care was taken to ensurethat no gas was allowed to enter into or escape from the reactor.

HPLC:

HPLC System Agilent 1100 Series. Mobile Phase: 0.0025N Sulfuric Acid.Flow and pressure: 0.800 mL/min. Column: Alltech IOA; Catalog #9648,150×6.5 mm, particle size 5 μm. Temperature of column: 60° C. Detector:Refractive Index. Temperature of detector: 45° C.

Method for Sample Preparation:

400 μL of sample and 50 μL of 0.15M ZnSO₄ and 50 μL of 0.15M Ba(OH)₂ areloaded into an Eppendorf tube. The tubes are centrifuged for 10 min. at12,000 rpm, 4° C. 200 μL of the supernatant are transferred into an HPLCvial, and 5 μL are injected into the HPLC instrument.

Gas Chromatography:

Gas Chromatograph HP 5890 series II utilizing a Flame IonizationDetector. Capillary GC Column: EC1000-Alltech EC1000 30 m×0.25 mm×0.25um. The Gas Chromatograph was operated in Split mode with a total flowof hydrogen of 50 mL/min with 5 mL purge flow (1:10 split), a columnhead pressure of 10 PIS resulting in a linear velocity of 45 cm/sec. Thetemperature program was initiated at 60° C., held for 1 minute thenramped to 215° C. at 30° C. per minute, then held for 2 minutes.Injector temperature was 210° C. and the detector temperature was 225°C.

Method for Sample Preparation:

500 μL sample is centrifuged for 10 min at 12,000 rpm, 4° C. 100 μL ofthe supernatant is transferred into an GC vial containing 200 μL waterand 100 μL of internal standard spiking solution (10 g/L propan-1-ol, 5g/L iso-butyric acid, 135 mM hydrochloric acid). 1 μL of the solution isinjected into the GC instrument.

Cell Density:

Cell density was determined by counting bacterial cells in a definedaliquot of fermentation broth. Alternatively, the absorbance of thesamples was measured at 600 nm (spectrophotometer) and the dry massdetermined via calculation according to published procedures.

Example 1 Batch Fermentation in CSTR

1.5 litres of media solution A was aseptically and anaerobicallytransferred into a 2 L CSTR vessel, and continuously sparged with N₂.Once transferred to the fermentation vessel, the reduction state and pHof the transferred media could be measured directly via probes. Themedia was heated to 37° C. and stirred at 400 rpm and resazurin (2 g/L)was added. 2.25 ml of H3PO4 85% was added to obtain a 30 mM solution andthe pH was adjusted to 5.3 using NH4OH. The media was then reducedfurther to −150 mV by the addition of 0.3M Cr(II)chloride solution.

Polysulfide solution (4.5M) was added to the solution, and the pHadjusted to 5.5 using NH4OH. N₂ was continuously sparged through thesolution following the addition of the polysulfide solution. Metal ionswere added according to solution B and 15 ml of solution C was added.

Prior to inoculation, the gas was switched to a pre-mixed blend of 50%CO, 2% H₂, 19% CO2, and 29% N₂, which was continuously sparged into thefermentation broth throughout the experiment. An actively growingClostridium autoethanogenum culture was inoculated into the CSTR at alevel of approximately 5% (v/v). During these experiments, the pH wasadjusted and/or maintained by a controller through the automatedaddition of buffers (0.5 M NaOH or 2N H₂SO₄). Metabolite production andmicrobial growth can be seen in FIG. 1. FIG. 1 shows that typicalmetabolites including ethanol are produced throughout the microbialgrowth phase. In addition, lactate is produced through fermentation ofCO by Clostridium autoethanogenum. However, lactate is produced by themicrobial culture when substrate is supplied such that the specificuptake is maintained above 0.5 mmol/g biomass/minute.

Example 2 Batch Fermentation in CSTR

1.5 litres of the media solution A was aseptically and anaerobicallytransferred into a 2 L CSTR vessel, and continuously sparged with N₂.Once transferred to the fermentation vessel, the reduction state and pHof the transferred media could be measured directly via probes. Themedia was heated to 37° C. and stirred at 400 rpm and resazurin (2 g/L)was added. 2.025 ml of H3PO4 85% was added to obtain a 30 mM solutionand the pH was adjusted to 5.3 using NH4OH. The media was then reducedfurther to −150 mV by the addition of 0.3M Cr(II)chloride solution.

Polysulfide solution (3.5M) was added to the solution. N₂ wascontinuously sparged through the solution following the addition of thepolysulfide solution. Prior to inoculation, the gas was switched to apre-mixed blend of 54% CO, 3% H₂, 20% CO2, and 23% N₂, which wascontinuously sparged into the fermentation broth throughout theexperiment. 150 ml of solution D was added and 13.5 ml of solution C wasadded. An actively growing Clostridium autoethanogenum culture wasinoculated into the CSTR at a level of approximately 5% (v/v). Duringthese experiments, the pH was adjusted and/or maintained by a controllerthrough the automated addition of buffers (0.5 M NaOH or 2N H₂SO₄).

Metabolite production and microbial growth can be seen in FIG. 2. FIG. 2shows that typical metabolites including ethanol are produced throughoutthe microbial growth phase. However, lactate is produced by themicrobial culture when substrate is supplied such that the specificuptake is maintained above 0.5 mmol/g biomass/minute. Betweenapproximately day 1.0 and 1.5, the rate of lactate production was atleast 1.0 g/L/day.

Example 3 Continuous Fermentation in CSTR

1 litre of the media solution was aseptically and anaerobicallytransferred into a 1 L CSTR vessel, and continuously sparged with N₂.Once transferred to the fermentation vessel, the reduction state and pHof the transferred media could be measured directly via probes. Themedia was heated to 37° C. and stirred at 400 rpm. 1.2 ml of H3PO4 85%was added to obtain a 30 mM solution and the pH was adjusted to 5.3using NH4OH. 8 ml of solution C was added. The media was then reducedfurther to −150 mV by the addition of 0.3M Cr (II) chloride solution.Resazurin was then added (2 g/L).

Polysulfide solution (6M) was added to the solution. N₂ was continuouslysparged through the solution following the addition of the polysulfidesolution. Prior to inoculation, the gas was switched to a pre-mixedblend of 70% CO, 1% H₂, 15% CO2, and 14% N₂, which was continuouslysparged into the fermentation broth throughout the experiment. 80 ml ofsolution E was added. An actively growing Clostridium autoethanogenumculture was inoculated into the CSTR at a level of approximately 5%(v/v). During these experiments, the pH was adjusted and/or maintainedby a controller through the automated addition of buffers (0.5 M NaOH or2N H₂SO₄).

Metabolite production and microbial growth can be seen in FIG. 3. FIG. 3shows that typical metabolites including ethanol are produced throughoutthe microbial growth phase. However, lactate is produced by themicrobial culture when substrate is supplied such that the specificuptake is maintained above 0.5 mmol/g biomass/minute. The microbialculture continues to produce lactate even when growth and ethanolproduction. However, lactate production ceases when the specific uptakedrops below 0.5 mmol/g/min.

Example 4 Continuous Fermentation in CSTR

1.5 litres of media solution A having a MgCl₂.6H₂O content of 0.407 gwas aseptically and anaerobically transferred into a 2 L CSTR vessel,and continuously sparged with N₂. Once transferred to the fermentationvessel, the reduction state and pH of the transferred media could bemeasured directly via probes. The media was heated to 37° C. and stirredat 400 rpm and resazurin (2 g/L) was added. 0.56 ml of H3PO4 85% wasadded to obtain a 5 mM solution and the pH was adjusted to 5.3 usingNH4OH.

Iron (0.1M), Nickel (0.05M) and Zinc (0.005M) were added to thesolution, as was 0.01M (B, Mn, Co, Se, Mo) and 0.01M Tungsten solution.15 ml of B-Vitamin was also added. The media was then reduced further to−200 mV by the addition of 0.3M Cr(II)chloride solution

Prior to inoculation, the gas was switched to a pre-mixed blend of 10%CO, 15% H₂, 75% RMG, which was continuously sparged into thefermentation broth throughout the experiment. An actively growingClostridium autoethanogenum culture was inoculated into the CSTR at alevel of approximately 5% (v/v). During these experiments, the pH wasadjusted and/or maintained by a controller through the automatedaddition of buffers (0.5 M NaOH or 2N H₂SO₄).

Metabolite production and microbial growth can be seen in FIG. 4. FIG. 4shows that typical metabolites including ethanol are produced throughoutthe microbial growth phase. However, lactate is produced by themicrobial culture when substrate is supplied such that the specificuptake is maintained above 0.6 mmol/g biomass/minute.

Example 5 Effect of Lactic Acid Concentration on Growth and MetaboliteProduction

The effect of lactic acid concentrations on acetate production andbiomass was examined using serum bottles. One litre of Solution F wasmade with an addition of 1 g/l of yeast extract. The media was splitinto five aliquots of 200 ml, and to each, DL-Lactic acid was added to adifferent final concentration. This was followed by pH adjustment to 5.5using 5M NaOH. The concentrations tested were 0 g/l, 0.3 g/l, 0.6 g/l, 1g/l and 5 g/l.

The media was bubbled with N2 for at least one hour and dispensed intoserum bottles in 50 ml aliquots under anaerobic conditions. Serumbottles were autoclaved for 30 mins at 121° C.

A microbial culture comprising C. autoethanogenum was grown in theprepared media under a mill gas headspace (30 psig) at 37° C. for threedays or until exponential growth is reached (OD600 approximately 0.5)

The serum bottles were inoculated with 2.5 ml of culture, pressurised to30 psig with mill gas and incubated at 37° C., shaking. OD600 change andmetabolite production were monitored through daily sampling.

The experimental setup was carried out in a similar fashion as discussedabove, using range of lactic acid concentrations. The concentrationstested were 0 g/l, 1 g/l, 2 g/l, 3 g/l, 4 g/l and 20 g/l.

The cultures were then monitored over seven (7) days to determine thegrowth of the culture in biomass, the amount of acetate produced by theculture and the amount of Lactic acid taken up by the culture.

At concentrations of between 0.3 g/l and 1 g/l the amount of biomassobserved was comparable to the culture with no lactic acid.Concentrations of between 2 g/l and 4 g/l showed reduced biomass whencompared to zero concentration lactic acid, and no growth was observedin cultures comprising 5 g/l or more of lactic acid.

Correspondingly, the serum bottles having a lactic acid concentration of5 g/l or greater demonstrated no acetate production and no lactic aciduptake.

The invention has been described herein with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. Those skilled in the art willappreciate that the invention is susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the invention includes all such variations andmodifications. Furthermore, titles, headings, or the like are providedto enhance the reader's comprehension of this document, and should notbe read as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications,cited above and below, if any, are hereby incorporated by reference.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in the UnitedStates of America or any country in the world.

Throughout this specification and any claims which follow, unless thecontext requires otherwise, the words “comprise”, “comprising” and thelike, are to be construed in an inclusive sense as opposed to anexclusive sense, that is to say, in the sense of “including, but notlimited to”.

1-21. (canceled)
 22. A method of producing lactate by microbialfermentation, said method comprising: providing a substrate thatincludes CO; in a bioreactor containing a culture of one or moremicro-organisms, anaerobically fermenting the substrate to producelactate.
 23. The method of claim 22, wherein providing the substratecomprises providing the substrate at a level sufficient to producelactate.
 24. The method of claim 22 wherein providing the substratecomprises providing the substrate at a level sufficient to maintain aspecific CO uptake rate of at least 0.4 mmol CO/gram dry cells weight ofbacteria/minute by the culture.
 25. The method of claim 24, whereinproviding the substrate comprises providing the substrate at a levelsufficient to maintain a specific CO uptake rate of at least 0.6 mmolCO/g/min.
 26. The method of claim 22 wherein one or more of the one ormore micro-organisms includes one or more lactate dehydrogenase genes,and wherein the method further includes upregulating the lactatedehydrogenase gene(s), such that lactate is produced by themicro-organism(s).
 27. The method of claim 26, wherein providing thesubstrate comprises providing the substrate at a level sufficient toproduce lactate.
 28. The method of claim 26, wherein providing asubstrate that includes CO comprises providing the substrate at a levelsufficient to maintain a specific rate of CO uptake of at least 0.4 mmolCO/gram dry cells weight of bacteria/minute by the culture.
 29. Themethod of claim 22 wherein the one or more micro-organism(s) compriseClostridium autoethanogenum.
 30. The method of claim 22 whereinproviding a substrate that contains CO comprises providing a gaseoussubstrate.
 31. The method of claim 30, wherein providing a gaseoussubstrate comprises providing a gaseous substrate that contains at leastabout 15% to about 100% CO by volume.
 32. The method of claim 30,wherein providing a gaseous substrate that includes CO comprisesproviding gas obtained as a by-product from an industrial process.
 33. Amethod of producing lactate by microbial fermentation, the methodincluding: providing a substrate in a bioreactor that contains a culturehaving Clostridium autoethanogenum, anaerobically fermenting thesubstrate to produce lactate.
 34. The method of claim 33, whereinproviding a substrate comprises providing a substrate that includes oneor more carbohydrates.
 35. The method of claim 34, wherein the methodfurther comprises providing a substrate that includes CO before, at thesame time, or after the step of providing a substrate comprising one ormore carbohydrates.
 36. The method of claim 35, wherein the methodfurther comprises alternating between providing a substrate comprisingCO and providing a substrate comprising one or more carbohydrates.